Loss of Tafazzin (TAZ) Function and Accelerated Apoptosis of Human Bone Marrow Stem and Myeloid Progenitors in Barth Syndrome.

Abstract 549

Barth syndrome (BTHS) is a severe X-linked stem cell disorder characterized by neutropenia, cardio- and skeletal myopathies, and growth retardation. Barth patients have a high rate of mortality due to progressive cardiomyopathy and/or overwhelming bacterial infections. Majority of Barth patients have mutations in the tafazzin (G4.5 or TAZ) gene that appear to truncate the tafazzin protein resulting in the loss of TAZ function. Based on protein homology, tafazzin is a phospholipid acyltransferase involved in remodeling cardiolipin (CL), the main lipid of the inner mitochondrial membrane. Therefore, BTHS patients exhibit reduced levels of total CL and accumulation of monolysocardiolipin. However, the function of TAZ protein and how these metabolic defects are triggered by TAZ mutations in BTHS remain largely unknown. The cellular or mouse models of this disorder are not availabel yet, and thus, the link between TAZ mutations and severe neutropenia in Barth syndrome remains elusive. Earlier study reported increased annexin V staining but absence of apoptosis in peripheral blood neutrophils. However, the patients' bone marrow stem and myeloid progenitor cells have not been examined. We hypothesized that TAZ mutations trigger accelerated apoptosis of bone marrow stem/progenitor cells, which in turn leads to reduced production of neutrophils in the bone marrow and severe neutropenia in Barth patients. To test this hypothesis, we used TAZ-specific shRNA to knock down the expression of the tafazzin gene in human myeloid progenitor HL60 cells and examined its effect on cell survival. Transfection of human myeloid progenitor cells with two different TAZ-specific but not control scrambled shRNA results in substantial down-regulation in the tafazzin expression level as determined by Western blot and confirmed by RT-PCR using TAZ and GAPDH-specific primers. Human myeloid progenitor cells with knocked down TAZ expression exhibit significantly elevated dissipation of mitochondrial membrane potential compared with control cells with scrambled shRNA as evidenced by flow cytometry analysis of DIOC6-labeled cells ((p<0.0009, n=3). A remarkably significant increase in proportion of apoptotic annexin-positive cells was also observed in response to knock-down of TAZ expression in myeloid progenitor cells compared with control cells with scrambled shRNA (p<0.0002, n=6). The observed increase in apoptosis in response to TAZ knock-down was caspase-3 dependent as evidenced by Western blot analysis. Treatment of the cells with caspase-specific inhibitor zVAD-fmk significantly improved cell survival characteristics to near normal level as determined by flow cytometry (p<0.02, n=4). Interestingly, knock-down of TAZ expression in human lymphoid cells failed to affect their cell survival or mitochondrial membrane potential, indicating that the loss of TAZ function exhibits lineage-specific effect. Analysis of bone marrow-derived CD34+ stem cells from a Barth patient positive for TAZ mutation cultured 24h in the presence of 10% autologous serum revealed nearly 3-fold increase in proportion of apoptotic annexin V positive CD34+ cells compared with the same cell subpopulation from a healthy volunteer (36% in Barth vs 13% in ctrl). More differentiated CD15+ neutrophil precursors also exhibit substantially increased rate of apoptosis compared with control (Barth 27% vs ctrl 17%). CD33+ myeloid-committed progenitor cells from Barth patient do not show increased annexin V binding compared with corresponding cell subpopulation from a healthy volunteer, but demonstrate approximately 2-fold increase in dissipation of mitochondrial membrane potential compared with control donor cells. Thus, these data demonstrate that tafazzin functions as an anti-apoptotic protein, the loss of function of the TAZ gene is cytotoxic to hematopoietic cells, and that severe neutropenia in patients with Barth syndrome is due to accelerated apoptosis of bone marrow myeloid progenitor cells. Our data also reveal that caspase-specific inhibitors may represent potentially therapeutic agents capable of restoring normal production of myeloid cells in Barth Syndrome.